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Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.

ABSTRACT: Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.

SUBMITTER: Gitlin G

PROVIDER: S-EPMC1135399 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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Studies on the biotin-binding site of avidin. Tryptophan residues involved in the active site.

Project description:Egg-white avidin was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl bromide. The complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per avidin subunit. The identity of the modified residues was determined by isolating the relevant tryptic and chymotryptic peptides from CNBr-cleaved avidin fragments. The results demonstrate that Trp-70 and Trp-110 are modified in approximately equivalent proportions. It is believed that these residues are located in the active site of avidin and take part in the binding of biotin.

| S-EPMC1148846 | biostudies-other

Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

Project description:The involvement of tyrosine in the biotin-binding sites of the egg-white glycoprotein avidin and the bacterial protein streptavidin was examined by using the tyrosine-specific reagent p-nitrobenzenesulphonyl fluoride (Nbs-F). Modification of an average of about 0.5 mol of tyrosine residue/mol of avidin subunit caused the complete loss of biotin binding. This indicates that the single tyrosine residue (Tyr-33) in the avidin subunit is directly involved in the biotin-binding site and that its modification by Nbs also abolishes the binding properties of a neighbouring subunit. This suggests that the tyrosine residues of the egg-white protein may also contribute to the stabilization of the native protein structure. In streptavidin, however, the modification of an average of 3 mol of tyrosine residue/mol of subunit was required to inactivate completely the biotin-binding activity of the protein, but only 1 mol (average) of tyrosine residue/mol of subunit was protected in the presence of biotin. The difference between the h.p.l.c. elution profiles of the enzymic digests of Nbs-modified streptavidin and the Nbs-modified streptavidin-biotin complex revealed two additional fractions in the unprotected protein that contain Nbs-modified tyrosine residues. These residues, Tyr-43 (major fraction) and Tyr-54 (minor fraction), appear to contribute to the biotin-binding site in streptavidin.

| S-EPMC1131609 | biostudies-other

Studies on the biotin-binding site of avidin. Lysine residues involved in the active site.

Project description:Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

| S-EPMC1147797 | biostudies-other

A monovalent streptavidin with a single femtomolar biotin binding site.

Project description:Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

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A Streptavidin Binding Site Mutation Yields an Unexpected Result: An Ionized Asp128 Residue Is Not Essential for Strong Biotin Binding.

Project description:We report a detailed study of a point mutation of the crucial binding site residue, D128, in the biotin-streptavidin complex. The conservative substitution, D128N, preserves the detailed structure observed for the wild-type complex but has an only minimal impact on biotin binding, even though previous experimental and computational studies suggested that a charged D128 residue was crucial for high-affinity binding. These results show clearly that the fundamental basis for streptavidin's extremely strong biotin binding affinity is more complex than assumed and illustrate some of the challenges that may arise when analyzing extremely strong ligand-protein binding interactions.

| S-EPMC5030189 | biostudies-literature

Target-activated streptavidin-biotin controlled binding probe.

Project description:Target-activated chemical probes are important tools in basic biological research and medical diagnosis for monitoring enzyme activities and reactive small molecules. Based on the fluorescence turn-on mechanism, they can be divided into two classes: dye-based fluorescent probes and caged-luciferin. In this paper, we introduce a new type of chemical probe in which the fluorescence turn-on is based on controlled streptavidin-biotin binding. Compared to conventional probes, the streptavidin-biotin controlled binding probe has several advantages, such as minimal background at its "OFF" state, multiple signal amplification steps, and unlimited selection of the optimal dyes for detection. To expand the scope, a new synthetic method was developed, through which a wider range of analyte recognition groups can be easily introduced to construct the binding probe. This probe design was successfully applied to image and study secreted peroxynitrite (ONOO-) at the cell surface of macrophages where information on ONOO- is difficult to obtain. As the signals are generated upon the binding of streptavidin to the biotin probe, this highly versatile design can not only be used in fluorescence detection but can also be applied in various other detection modes, such as electrochemical and enzyme-amplified luminescence detection.

| S-EPMC5872805 | biostudies-other

Biotin Binding Hardly Affects Electron Transport Efficiency across Streptavidin Solid-State Junctions.

Project description:The electron transport (ETp) efficiency of solid-state protein-mediated junctions is highly influenced by the presence of electron-rich organic cofactors or transition metal ions. Hence, we chose to investigate an interesting cofactor-free non-redox protein, streptavidin (STV), which has unmatched strong binding affinity for an organic small-molecule ligand, biotin, which lacks any electron-rich features. We describe for the first time meso-scale ETp via electrical junctions of STV monolayers and focus on the question of whether the rate of ETp across both native and thiolated STV monolayers is influenced by ligand binding, a process that we show to cause some structural conformation changes in the STV monolayers. Au nanowire-electrode-protein monolayer-microelectrode junctions, fabricated by modifying an earlier procedure to improve the yields of usable junctions, were employed for ETp measurements. Our results on compactly integrated, dense, uniform, ∼3 nm thick STV monolayers indicate that, notwithstanding the slight structural changes in the STV monolayers upon biotin binding, there is no statistically significant conductance change between the free STV and that bound to biotin. The ETp temperature (T) dependence over the 80-300 K range is very small but with an unusual, slightly negative (metallic-like) dependence toward room temperature. Such dependence can be accounted for by the reversible structural shrinkage of the STV at temperatures below 160 K.

| S-EPMC9893813 | biostudies-literature

Mirror-image streptavidin with specific binding to L-biotin, the unnatural enantiomer.

Project description:The streptavidin-biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin-biotin system, even for in vivo use.

| S-EPMC9187662 | biostudies-literature

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

Project description:We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.

| S-EPMC1138535 | biostudies-other

Studies on the biotin-binding site of avidin. Minimized fragments that bind biotin.

Project description:The object of this study was to define minimized biotin-binding fragments, or 'prorecognition sites', of either the egg-white glycoprotein avidin or its bacterial analogue streptavidin. Because of the extreme stability to enzymic hydrolysis, fragments of avidin were prepared by chemical means and examined for their individual biotin-binding capacity. Treatment of avidin with hydroxylamine was shown to result in new cleavage sites in addition to the known Asn-Gly cleavage site (position 88-89 in avidin). Notably, the Asn-Glu and Asp-Lys peptide bonds (positions 42-43 and 57-58 respectively) were readily cleaved; in addition, lesser levels of hydrolysis of the Gln-Pro (61-62) and Asn-Asp (12-13 and 104-105) bonds could be detected. The smallest biotin-binding peptide fragment, derived from hydroxylamine cleavage of either native or non-glycosylated avidin, was identified to comprise residues 1-42. CNBr cleavage resulted in a 78-amino acid-residue fragment (residues 19-96) that still retained activity. The data ascribe an important biotin-binding function to the overlapping region (residues 19-42) of avidin, which bears the single tyrosine moiety. This contention was corroborated by synthesizing a tridecapeptide corresponding to residues 26-38 of avidin; this peptide was shown to recognize biotin. Streptavidin was not susceptible to either enzymic or chemical cleavage methods used in this work. The approach taken in this study enabled the experimental distinction between the chemical and structural elements of the binding site. The capacity to assign biotin-binding activity to the tyrosine-containing domain of avidin underscores its primary chemical contribution to the binding of biotin by avidin.

| S-EPMC1151383 | biostudies-other

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